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US5891679A - TNF-alpha muteins and a process for preparing them - Google Patents

TNF-alpha muteins and a process for preparing them Download PDF

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US5891679A
US5891679A US08/500,860 US50086095A US5891679A US 5891679 A US5891679 A US 5891679A US 50086095 A US50086095 A US 50086095A US 5891679 A US5891679 A US 5891679A
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tnf
amino acid
mtnf
activity
mutein
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Rudolph Lucas
Patrick De Baetselier
Lucia Fransen
Erwin Sablon
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Fujirebio Europe NV SA
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Innogenetics NV SA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to TNF- ⁇ -muteins, a process for preparing them and their use as active substances in pharmaceutical compositions.
  • Tumor necrosis factor- ⁇ also known as cachectin
  • TNF- ⁇ Tumor necrosis factor- ⁇
  • cachectin Tumor necrosis factor- ⁇
  • Tumor necrosis factor- ⁇ also known as cachectin
  • Tumor necrosis factor- ⁇ is a multifunctional cytokine produced mainly by activated macrophages. In vitro, it has diverse biological effects (reviewed by Manogue et Cerami, 1988) including killing of transformed cells (Carswell et al., 1975), stimulation of granulocytes and fibroblasts (Old, 1985; Vilchek et al., 1986; Beutler and Cerami, 1987), damage to endothelial cells (Sato et al, 1986), and anti-parasitic effects (Taverne et al., 1984).
  • TNF- ⁇ In vivo, it plays a key role as an endogenous mediator of inflammatory, immune and host defence functions and it is involved in a number of pathological conditions in man and mouse such as septic shock, cachexia, capillary leak syndrome, hemorhagic necrosis of multiple organs, etc. It is capable of acting independently and in conjunction with other factors affecting a whole plethora of different body functions. These effects can either be beneficial or life-threatening to the host. Some of these effects are direct, others may be mediated via the induction of other secreted factors. The biological effects of TNF- ⁇ are mediated via binding to specific cell surface receptors.
  • TNF- ⁇ human and mouse TNF- ⁇ (Pennica et al., 1984; Fransen et al., 1985) and of two different TNF-receptors (p55-TNF-R and p75-TNF-R) has been deduced from the nucleotide sequence of the cloned cDNA (Dembic et al., 1990; Loetscher et al., 1990). Both receptors bind not only TNF- ⁇ but also TNF- ⁇ or lymphotoxin with high affinity (Schoenfeld et al., 1991).
  • TNF- ⁇ is a related lymphocyte product (TNF- ⁇ and TNF- ⁇ share 32% homology on the amino acid sequence level) that exhibits pleiotropic activities very similar to those of TNF- ⁇ .
  • X-ray crystallographic analysis revealed that the tertiary structure of both molecules is virtually identical except that the TNF- ⁇ trimer creates a molecule that is less elongated than the TNF- ⁇ trimer and the latter has a top-region that flares open (Eck et al, 1992).
  • TNF- ⁇ has been shown to have a lectin-like property for the oligosaccharide ligands chitobiose and Man( ⁇ 1,3)-(Man( ⁇ 1,6)-Man (Hession et al., 1987; Sherblom et al., 1988). They further demonstrated that the TNF- ⁇ protein has at least two different binding sites, one lectin-like and the other directed at cell surface receptors.
  • TNF- ⁇ mutants were described for which the binding to the cellular TNF-receptor p55 and/or p75 was hampered. All these mutations are located in the lower half of the pyramidal structure of the biologically active TNF- ⁇ trimer (Van Ostade et al., 1991; Van Ostade et al., 1992; EP-A-0 486 908).
  • TNF- ⁇ tumor necrosis factor- ⁇
  • monoclonal antibodies as well as fragments thereof, which neutralize the in vitro and in vivo toxic properties of TNF- ⁇ (e.g. EP-A-0 350 690).
  • TNF- ⁇ has also been shown to be involved in the control of growth and differentiation of various parasites.
  • parasites Upon infection of the host, parasites are capable of inducing the secretion of different cytokines such as TNF which may affect the course of the disease.
  • TNF- ⁇ can be protective in certain circumstances, such as inhibiting parasite survival in rodent malaria (Clark et al., 1987; Taverne et al., 1987).
  • its overproduction can be detrimental to the host and can contribute to the pathology of the disease (Clark, 1987; Grau et al., 1989).
  • TNF- ⁇ a In schistosomiasis, the parasite uses the host-derived immunoregulatory protein TNF- ⁇ as a signal for replication and transmission (Amiri et al., 1992). Moreover, the authors found that the parasite worms required TNF- ⁇ for egg laying and for excretion of eggs from the host. In cutaneous leishmaniasis, TNF- ⁇ a plays a protective role (Titus et al., 1989; Liew et al., 1990).
  • African trypanosome species are the etiological agents for African trypanosomiasis, or sleeping sickness, and are a major cause of both human disease (Trypanosoma brucei rhodesiense and T. brucei gambiense) and cattle disease (T. brucei brucei, T. evansi, T. vivax, T. congolense) in Africa. Injected into their mammalian hosts by tsetse flies, trypanosomes remain extracellular bloodstream trypomastigotes throughout their mammalian phase.
  • TNF- ⁇ is the principle mediator of endotoxic or septic shock (Cerami and Beutler, 1988).
  • LPS endotoxin
  • TNF- ⁇ produces hypotension, leukopenia, and local tissue necrosis (Okusawa et al., 1988).
  • TNF-induced septic shock refers to the shock that develops in the presence of severe infection, especially following bacteremia with Gram-negative bacteria and release of endotoxin. It may also be caused by any class of microorganism, including Gram-positive bacteria, viruses, fungi, protozoa, spirochetes and rickettsiae.
  • the pathophysiology of septic shock is very complex (see Harrison's Principles of Internal Medicine, 12th ed, McGraw-Hill, New York, pp. 502-507). Death is caused by progression to multiple organ failure and circulatory collapse.
  • TNF- ⁇ has a lectin-like affinity for the glycoprotein uromodulin.
  • this glycoprotein is able to potently inhibit the TNF-induced shock in vivo, whereas it does not block the tumoricidal activity of TNF- ⁇ on L929 cells in vitro.
  • the aim of the present invention is to provide TNF- ⁇ muteins which have altered lectin-like properties in comparison with those of TNF- ⁇ , and/or TNF- ⁇ muteins which have a reduced toxic activity in comparison to TNF- ⁇ , and/or TNF- ⁇ muteins which have altered inflammatory cytokines inducing capacities with respect to TNF- ⁇ , and/or TNF- ⁇ muteins with reduced metastasis promoting activities with respect to TNF- ⁇ , and/or TNF- ⁇ muteins which have an extended half-life time with respect to TNF- ⁇ .
  • the aim of the present invention is to provide TNF- ⁇ muteins which have reduced lectin-like properties in comparison with those of TNF- ⁇ , and/or result in reduced cachexia or septic shock upon treatment with toxic concentrations of TNF- ⁇ in cytokine therapy, without having lost,their tumoricidal activity.
  • Another aim of the present invention is to provide pharmaceutical compositions comprising said TNF- ⁇ muteins.
  • the aim of the present invention is also to provide antisense peptides or antibodies which recognize the TNF- ⁇ lectin-like epitope for use in the preparation of a medicament for treating endogenous toxic concentrations of TNF- ⁇ due to sepsis, septic shock, Gram negative sepsis, endotoxic shock, toxic shock syndrome, cachexia, microbial infections, rheumatoid arthritis, inflammatory conditions, respiratory distress syndrome, pulmonary fibrosis, infections, graft-versus-host-disease, reperfusion damage such as myocardial ischaemia, AIDS, cancer, cerebral malaria, immunosuppression, etc.
  • Another aim of the present invention is to use said antibodies or said antisense peptides, in addition to complete TNF- ⁇ , for the preparation of a medicament for treating tumors, with said medicament having reduced toxic side effects and/or altered (i.e. reduced or increased) lectin-like effects and/or altered inflammatory cytokines inducing capacities, and/or altered adhesion molecules inducing capacities, and/or an increased half-life with respect to TNF- ⁇ in the absence of said antibodies or antisense peptides.
  • the present invention relates more particularly to a tumor necrosis factor mutein characterized in that the TNF- ⁇ amino acid sequence is mutated, or deleted totally or partially, in the region extending from amino acid position at 101 to 116 in such a way that:
  • TNF- ⁇ muteins have preferentially retained the tumoricidal activity of TNF- ⁇ , and providing that said TNF muteins are different from human TNF- ⁇ wherein amino acids 1 to 8 are replaced by a sequence within the region spanning amino acids 5 to 30 of laminin, and providing that said TNF muteins are different from human TNF- ⁇ wherein amino acid position 101 is Ser, hTNF- ⁇ wherein amino acid position 102 is Arg or deleted, hTNF- ⁇ wherein amino acid position 103 is Trp, hTNF- ⁇ wherein amino acid position 105 is Pro, hTNF- ⁇ wherein amino acid position 105 is Ile, hTNF- ⁇ wherein amino acid position 105 is Ile and position 44 is Cys, hTNF- ⁇ wherein amino acid position 106 is Ser, hTNF- ⁇ wherein amino acid position 106 is Ser and position 131 is Cys, hTNF- ⁇
  • TNF muteins possibly containing in their peptidic chain outside the region spanning amino acids 101 to 116 of TNF- ⁇ , additional modifications consisting of substitutions and/or deletions and/or additions of one or several amino acid residues, and with said muteins being characterized in that they have retained the aforementioned activities; or a pharmaceutically acceptable salt thereof.
  • lectin-like activities which are modulated (i.e. reduced or increased), preferably reduced, from 5 to 100%, preferably at least 25%, more preferably at least 50%, most preferably at least 75%, as compared with lectin-like activities observed for TNF- ⁇ ; or,
  • inflammatory cytokines inducing capacities which are modulated (i.e. either reduced or increased), preferably reduced, from 5 to 100%, preferably at least 25%, more preferably at least 50%, most preferably at least 75% with respect to TNF- ⁇ ; or,
  • adhesion molecules inducing capacities which are modulated (i.e. either reduced or increased), preferably reduced, from 5 to 100%, preferably at least 25%, more preferably at least 50%, most preferably at least 75% with respect to TNF- ⁇ ; or,
  • metastasis promoting activity which is reduced from 5 to 100%, preferably at least 25%, more preferably at least 50%, most preferably at least 75% with respect to TNF- ⁇ ; or,
  • TNF- ⁇ tumoricidal activity of TNF- ⁇ being preferentially retained by said muteins.
  • TNF- ⁇ muteins have a tumoricidal activity of at least 75%, preferably at least 85%, more preferably at least 95% and even more preferably at least 100% of the tumoricidal activity of TNF- ⁇ which has not been mutated in the region spanning amino acids 101 to 116. It is, however, also possible that TNF-muteins according to the present invention have largely lost the tumoricidal activities of TNF- ⁇ , whilst in addition having at least one, or preferably a combination, of any of the aforementioned activities of the TNF muteins according to the invention which are modulated (i.e. increased or reduced).
  • TNF-muteins which have increased lectin-like activities and/or a reduced toxicity, and/or a reduced metastasis promoting activity, and/or an extended half life, and which can no longer be classified as having a tumoricidal activity are also contemplated within the present invention.
  • TNF-muteins according to the present invention will mostly show a combination of at least two of the aforementioned effects. Illustrations of preferred TNF-muteins according to the present invention are given below and in the Examples section.
  • toxic activity refers to the in vivo TNF- or LPS-induced septic shock effect of TNF- ⁇ as explained above, and may be measured in vivo on the basis of the increased survival time of C57BL/6 mice, upon injection with fixed dosages of the TNF- ⁇ mutein, as compared with that in C57BL/6 mice injected with the same dosages of unmodified TNF- ⁇ ; or by any other technique known in the art.
  • tumoricidal activity refers to the in vivo tumor cell killing activity of TNF- ⁇ , which may be measured by means of any assay comprised in the art.
  • cytotoxic or cytolytical activity refers to the in vitro tumor cell killing activity of TNF- ⁇ .
  • an in vitro assay is reported to measure the cytotoxic activity of TNF- ⁇ on L929 TNF-sensitive fibrosarcoma cells.
  • An in vivo assay to evaluate the tumoricidal activity of TNF- ⁇ on HT-29 tumors in nude mice, and by means of the B16B16 melanoma test system, are also reported in the Examples section.
  • the term "lectin-like activities" of the present invention refers to the lectin-like properties of TNF- ⁇ .
  • the lectin-like properties of TNF- ⁇ are for instance those described by Hession et al. (1988) and Sherblom et al. (1988) and include any biological effect mediated by any lectin-carbohydrate interaction located in the region extending from amino acids 101 to 116 of TNF- ⁇ .
  • the "lectin-like activities" of the TNF- ⁇ muteins of the invention may be measured as the percentage of inhibition of trypanocidal activity of the TNF mutein, upon prior incubation of said TNF mutein with the oligosaccharide sugar N,N'-diacetylchitobiose as described in the Examples section; or by means of any other assay comprised in the art.
  • trypanocidal activity refers to the trypanocidal activity of TNF- ⁇ located in the region extending from amino acid positions 101 to 116 of TNF- ⁇ may be measured by means of an in vitro or an in vivo trypanocidal activity assay as extensively explained in the Examples section.
  • inflammatory cytokines inducing capacities refers to the stimulation capacity of TNF- ⁇ of cytokines such as IL1 ⁇ , IL1 ⁇ , IL8, TNF, IL6 and other molecules known to have a role in inflammatory processes by endothelial cells, said capacities being located in the region extending from amino acids 101 to 116 of TNF- ⁇ .
  • metastasis promoting activity within the meaning of the present invention refers to negative in vivo side effects of TNF- ⁇ exerted against tumor cells and resulting in a net increase/growth of the number of metastase such as reviewed by Orosz et al., 1993, located within the region extending from amino acid positions 101 to 116 of TNF ⁇ .
  • half-life refers to the in vivo measured time during which the TNF- ⁇ muteins of the invention remain in circulation and are detectable in body fluids.
  • TNF- ⁇ muteins on the inflammatory cytokines inducing capacities of TNF ⁇ may be measured in vivo by any technique known in the art, more particularly any of the techniques disclosed in the examples section of the present invention.
  • TNF- ⁇ muteins on the adhesion molecules inducing capacities of TNF- ⁇ may be measured in vivo by any technique known in the art.
  • TNF- ⁇ muteins The inhibition of metastasis promoting activity by these TNF- ⁇ muteins may be measured in vivo by any technique known in the art, or as disclosed in the examples section.
  • TNF- ⁇ muteins may be measured in vivo by any technique known in the art, or as disclosed in the examples section.
  • TNF- ⁇ refers to polypeptides of human origin containing 157 amino acid residues, as disclosed by Pennica et al. (1984), namely (SEQ ID NO: 35):
  • TNF- ⁇ also refers to polypeptides of mouse origin containing 156 amino acid residues, disclosed by Fransen et al. (1985) namely (SEQ ID NO: 36):
  • TNF- ⁇ amino acid sequence which is mutated in the region extending from amino acid position 101 to 116 refers to either deletions, insertions and/or substitutions in the region of amino acid positions 101 to 116 of TNF- ⁇ (including both positions 101 and 116).
  • the result of all of these deletions is such that at least one of the effects mentioned-above, and preferably combinations of at least two such effects of TNF- ⁇ , ascribed to the region covering amino acid positions 101 to 116 of TNF- ⁇ , are modulated and by preference are strongly reduced or abolished in the case of life-threatening lectin-like effects (toxic effect, inflammatory cytokines inducing activities, adhesion molecules inducing activities), metastasis promoting effects of TNF- ⁇ , or are by preference increased in the case of half-life times and beneficial lectin-like activities (such as induction of cytokines or adhesion molecules which are beneficial to the envisaged treatment).
  • life-threatening lectin-like effects toxic effect, inflammatory cytokines inducing activities, adhesion molecules inducing activities
  • metastasis promoting effects of TNF- ⁇ or are by preference increased in the case of half-life times and beneficial lectin-like activities (such as induction of cytokines or adhesion molecules which are beneficial to the
  • Table I gives an overview of the amino acid substitutions which could be the basis of some of the additional modifications in the TNF- ⁇ muteins in the region outside the region extending from amino acid positions 101 to 116 as defined above.
  • TNF- ⁇ -derived synthetic peptides located in the region extending from amino acid positions 101 to 116 of TNF- ⁇ as indicated in the sequences above, and which will be further referred to as the "TNF- ⁇ tip peptides", and which can be represented by the following general formula:
  • X1 can represent Pro, Ala, Thr, or Gly
  • X2 can represent Gly, Ala, Thr, Pro, or Ser;
  • Y2 can represent Lys or Arg
  • Y4 can represent Leu or Ala
  • TNF- ⁇ amino acid position numbering in the present application refers to the numbering as given in the sequences above.
  • region covering amino acids 101 to 116 of human TNF- ⁇ corresponds to amino acids 100 to 115 of mouse TNF- ⁇ , since in the sequences given above, a space has been introduced in the mouse TNF- ⁇ amino acid sequence position 73 in order to align it with the human TNF- ⁇ amino acid sequence.
  • the trypanocidal activity of the TNF- ⁇ "tip-region" can be inhibited by preincubation with N,N'-diacetylchitobiose, for which TNF- ⁇ has a lectin-like activity (Hession et al., 1987; Sherblom et al., 1988). Therefore, the tip region of TNF- ⁇ is shown to be implicated in the lectin-like activities of the cytokine.
  • the glycoprotein uromodulin which binds TNF- ⁇ via its lectin-like domain, can inhibit lipopolysacharide (LPS)-induced septic shock, caused by TNF- ⁇ , but can not inhibit the TNF- ⁇ tumoricidal effect on L929 cells, the lectin-like region of TNF- ⁇ , and consequently the tip-region of TNF- ⁇ , are shown by the present inventors to be implicated in the toxic side effects of the cytokine in vivo.
  • LPS lipopolysacharide
  • TNF- ⁇ muteins (extending from positions 101 to 116 of TNF- ⁇ ) were constructed in the tip-region which are characterized in that either the lectin-like activities are reduced with respect to TNF- ⁇ , or the toxic activity is reduced with respect to TNF- ⁇ ; or the half life is increased with respect to TNF- ⁇ ; or the metastasis promoting activities are reduced with respect to TNF- ⁇ , or the inflammatory cytokines inducing capacities are reduced with respect to TNF- ⁇ , or a combination of these effects are reduced/increased with respect to TNF- ⁇ and with said muteins being further characterized in that they have not lost the tumoricidal activity of TNF- ⁇ ; as detailed above.
  • the present invention relates more particularly to a TNF mutein as defined above, further characterized in that the lectin-like activities are modulated with respect to TNF- ⁇ .
  • the present invention relates to a TNF mutein as defined above, further characterized in that the lectin-like activities are reduced with respect to TNF- ⁇ .
  • the region spanning amino acids 101 to 116, and more particularly 105 to 110 is involved not only in the trypanocidal but also in the lectin-like activities of TNF- ⁇ .
  • the lectin-like activities were measured indirectly via the inhibition of the trypanocidal activity of TNF- ⁇ .
  • the present invention also relates to a TNF mutein as defined above, further characterized in that the lectin-like activities are increased with respect to TNF- ⁇ .
  • TNF muteins having increased lectin-like activities may be beneficial to certain envisaged treatments.
  • the present invention relates to a TNF mutein as defined above, further characterized in that the toxic activity is reduced with respect to TNF- ⁇ . It is illustrated in the examples section of the present invention that mutations to an Ala at positions T(105), E(107) and/or E(110) and a deletion of the region extending from amino acids 105 to 110 of mTNF ⁇ leads to mTNF- ⁇ muteins with a lower LD50 value as measured in vivo.
  • the present invention relates to a TNF mutein as defined above, further characterized in that the inflammatory cytokine inducing capacities are modulated with respect to TNF- ⁇ .
  • the present invention relates to a TNF mutein as defined above, further characterized in that the inflammatory cytokine inducing capacities are increased with respect to TNF- ⁇ .
  • mTNF- ⁇ muteins bearing a deletion of the region spanning amino acids 105-110 of mTNF- ⁇ show a higher induction of human interleukin-6 on human microvascular endothelial cells of the brain in vivo than wild-type mTNF ⁇ .
  • the present invention relates to a TNF mutein as defined above, further characterized in that the inflammatory cytokine inducing capacities are reduced with respect to TNF- ⁇ . It is shown in the examples section that T(105)A and P(106)A mTNF- ⁇ muteins as well a deletion mutant of mTNF ⁇ in the region extending from amino acids 105 to 110 induce consistently less human interleukin 8 (IL8) than wild-type TNF- ⁇ on human endothelial cells as measured in vivo.
  • IL8 human interleukin 8
  • the present invention relates to a TNF mutein as defined above, further characterized in that the adhesion molecule inducing capacities are modulated with respect to TNF- ⁇ .
  • the present invention relates to a TNF mutein as defined above, further characterized in that the adhesion molecule inducing capacities are reduced with respect to TNF- ⁇ .
  • the present invention refers to a TNF mutein as defined above, further characterized in that the adhesion molecule inducing capacities are increased with respect to TNF- ⁇ .
  • the present invention relates to a TNF mutein as defined above, further characterized in that the metastasis promoting activity is reduced with respect to TNF- ⁇ .
  • a mTNF ⁇ mutein carrying a deletion of the region extending from amino acids 105 to 110 shows a considerably lower in vivo metastasis enhancing potential compared to wild-type mTNF ⁇ in a murine lung carcinoma cell line model.
  • the present invention relates to a TNF mutein having at least one of the aforementioned effects, further characterized in that the tumoricidal activity is retained with respect to TNF- ⁇ .
  • TNF- ⁇ tumoricidal activity
  • any of the TNF muteins having at least one of the effects as defined above are further characterized in that their tumoricidal activity is reduced with respect to TNF- ⁇ .
  • the loss of the tumoricidal effect by certain TNF muteins of the invention may be desirable.
  • the present invention relates to a TNF mutein as defined above, further characterized in it shows an increased half life time with respect to TNF- ⁇ .
  • the tip deleted (amino acids 105-110) mTNF ⁇ muteins shows an increased half life as measured by means of the L929 cytotoxic assay and the indirect ELISA compared to wild-type mTNF ⁇ .
  • the present invention relates to any TNF mutein as defined above, characterized in that at least part of the region extending from amino acid positions 101 to 116 of TNF- ⁇ , or the complete region corresponding to amino acid positions 101 to 116 of TNF- ⁇ has been deleted, and preferably at least the region covering amino acid positions 105 to 110 has been deleted.
  • human TNF- ⁇ wherein amino acids 1 to 8 are replaced by a sequence within the region spanning amino acids 5 to 30 of laminin, and providing that said TNF muteins are different from human TNF- ⁇ wherein amino acid position 105 is Pro, hTNF- ⁇ wherein amino acid position 105 is Ile, hTNF- ⁇ wherein amino acid position 105 is Ile and position 44 is Cys, hTNF- ⁇ wherein amino acid position 106 is Ser, hTNF- ⁇ wherein amino acid position 106 is Ser and position 131 is Cys, hTNF- ⁇ wherein amino acid position 108 is Phe, hTNF- ⁇ wherein amino acid position 110 is Lys, and hTNF- ⁇ wherein amino acid positions 109 and 120 are respectively Gln and His and amino acid 111 is deleted, and may contain substitutions as set out in Table 1, in the region of TNF- ⁇ outside the region spanning from amino acid position 105 to amino acid position 110.
  • the present invention relates to TNF muteins wherein the region between amino acid positions 101 and 116 has been mutated in such a way that the amino acids Thr(105), Glu(107) and/or Glu(110) have been mutated to an Alanine residue, as demonstrated in the examples section.
  • TNF muteins wherein the region between amino acid positions 101 and 116 has been mutated in such a way that the amino acids Thr(105), Glu(107) and/or Glu(110) have been mutated to an Alanine residue, as demonstrated in the examples section.
  • mouse TNF- ⁇ muteins also human TNF- ⁇ muteins are contemplated within the present invention.
  • the present invention relates to a nucleic acid sequence encoding any of the TNF mutein polypeptides as defined above.
  • an appropriate cellular host with a vector, particularly a plasmid, a cosmid, a phage or a virus, in which a nucleic acid sequence as defined above coding for at least one of the TNF mutein polypeptides as defined above has been inserted (insert) under the control of the appropriate regulatory elements, particularly a promoter recognized by the polymerases of the cellular host and, in the case of a procaryotic host, an appropriate ribosome binding site (RBS), enabling the expression in said cellular host of said nucleic acid sequence,
  • a vector particularly a plasmid, a cosmid, a phage or a virus, in which a nucleic acid sequence as defined above coding for at least one of the TNF mutein polypeptides as defined above has been inserted (insert) under the control of the appropriate regulatory elements, particularly a promoter recognized by the polymerases of the cellular host and, in the case of a procaryotic
  • the TNF- ⁇ muteins according to the present invention may advantageously be used for the fact that they have a tumoricidal activity, while they lack either the life-threatening toxic, or the lectin-like properties of TNF- ⁇ , or the metastasis promoting effects of TNF- ⁇ , or the inflammation cytokines inducing capacities of TNF- ⁇ or mixture of these effects, and/or because they have an increased half life compared to TNF- ⁇ , and/or because they have increased beneficial lectin-like activities and/or inflammatory cytokines inducing activities, and/or ashesion molecules inducing activities compared to TNF- ⁇ .
  • amino acids 1 to 8 are replaced by a sequence within the region spanning amino acids 5 to 30 of laminin, and providing that said TNF muteins are different from human TNF- ⁇ wherein amino acid position 101 is Ser, hTNF- ⁇ wherein amino acid position 102 is Arg or deleted, hTNF- ⁇ wherein amino acid position 103 is Trp, hTNF- ⁇ wherein amino acid position 105 is Pro, hTNF- ⁇ wherein amino acid position 105 is Ile, hTNF- ⁇ wherein amino acid position 105 is Ile and position 44 is Cys, hTNF- ⁇ wherein amino acid position 106 is Ser, hTNF- ⁇ wherein amino acid position 106 is Ser and position 131 is Cys, hTNF- ⁇ wherein amino acid position 108 is Phe, hTNF- ⁇ wherein amino acid position 110 is Lys, hTNF- ⁇ wherein amino acid positions 111 to 112 are deleted, hTNF- ⁇ wherein amino acid position 112 is deleted or Met,
  • the TNF muteins used for treating the above-specified illnesses contain either a mutation of one or more amino acids or a deletion of at least part of the region spanning amino acids 105 to 110 of TNF- ⁇ . Particularly preferred examples as well as their biological effects are discussed in the examples section.
  • inflammatory cytokines inducing capacities are modulated with respect to TNf- ⁇
  • TNF- ⁇ muteins have preferentially retained the tumoricidal activity of TNF- ⁇ , and with said TNF muteins possibly containing in their peptidic chain outside amino acid region 101 to 116 (or respectively 105 to 110) of TNF- ⁇ , additional modifications consisting of substitutions and/or deletions and/or additions of one or several amino acid residues, and with said muteins being characterized in that they have retained the aforementioned activities; or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for treating illnesses and pathological conditions, such as, sepsis, septic shock, Gram negative sepsis, endotoxic shock, toxic shock syndrome, cachexia, microbial infections, rheumatoid arthritis, inflammatory conditions, respiratory distress syndrome, pulmonary fibrosis, infections, graft-versus-host-disease, reperfusion damage such as myocardial ischaemia, AIDS, cancer, immunosup
  • the TNF- ⁇ muteins according to the present invention may especially be used for the fact that they may have a tumoricidal activity, while they lack either the toxic, and/or the lectin-like properties of TNF- ⁇ , and/or the metastasis promoting effects of TNF- ⁇ , and/or the inflammatory cytokines inducing capacities of TNF ⁇ , and/or the adhesion molecules inducing capacities of TNF ⁇ , or mixture of these effects, and/or because they have an increased half life compared to TNF- ⁇ .
  • the present invention relates to the use of an antibody specifically detecting an epitope residing in the region encompassing amino acids 101 to 116 of TNF- ⁇ , preferably in the region encompassing amino acids 105 to 110 of TNF ⁇ more particularly a monoclonal antibody, characterized in that it:
  • TNF- ⁇ modulates (i.e. inhibit or stimulate), preferably inhibit, the lectin-like effects of TNF- ⁇ preferentially from 5 to 100%, preferably at least 25%, more preferably at least with 50%, most preferably with at least 75%; or,
  • the inhibition/stimulation of the lectin-like activities of TNF- ⁇ by the antibodies of the invention is measured as the percentage of inhibition of trypanocidal activity upon prior incubation of TNF- ⁇ with said antibody of the invention in the presence and/or absence of N,N'-diacetylchitobiose; or by any other method known in the art.
  • the monoclonal antibody of the invention should form an immunological complex with the subregion of the TNF- ⁇ "tip-region" responsible for either of the effects of TNF- ⁇ .
  • the present invention relates to the use of an immunological complex, comprising a monoclonal antibody as defined above, and TNF- ⁇ , for the preparation of a medicament.
  • a pharmaceutical composition containing, as active substance, an immunological complex comprising a monoclonal antibody as defined above, and complete TNF- ⁇ , in association with a pharmaceutical acceptable vehicle, is provided.
  • compositions could also contain IL-1 ⁇ , IL-1 ⁇ , IL-6, IFN-gamma, IFN ⁇ / ⁇ , IL-8, or any other immunoreactive agent.
  • composition can be used for treating illnesses wherein a treatment with TNF- ⁇ is beneficial such as tumors.
  • the monoclonal antibodies of the invention can be produced by any hybridoma liable to be formed according to classical methods from splenic cells of an animal, particularly of a mouse or rat, immunized against the polypeptides according to the invention, or muteins thereof, or fragments thereof defined above on the one hand, and of cells of a myeloma cell line on the other hand, and to be selected by the ability of the hybridoma to produce the monoclonal antibodies recognizing the polypeptides which has been initially used for the immunization of the animals.
  • the antibodies involved in the invention can be labelled by an appropriate label of the enzymatic, fluorescent, or radioactive type.
  • the monoclonal antibodies according to this preferred embodiment of the invention may be human monoclonal antibodies.
  • human monoclonal antibodies are prepared by EBV immortalization or, for instance, by means of human peripheral blood lymphocyte (PBL) repopulation of severe combined immune deficiency (SCID) mice (for recent review see Duchosal et al., 1992).
  • PBL peripheral blood lymphocyte
  • SCID severe combined immune deficiency
  • monoclonal antibodies, or fragments thereof can be modified for various uses, and fragments can be generated which retain the antigen binding properties.
  • fragments are commonly generated by, for instance enzymatic digestion of the antibodies with papain, pepsin, or other proteases.
  • the peptides according to this embodiment of the invention may be circularized before immunization.
  • the expression “circularized” refers to a joining of the N- and C- terminal cysteine residues by means of oxidation at low molar concentration to promote intramolecular disulfide bridges or by any other technique comprised in the art.
  • the peptides used for immunization are preferably in the form in which they are joined to a biotin molecule, or any other carrier molecule in order to achieve a good immunogenic response.
  • the biotin molecule may be coupled to the peptide of choice via a linker, such as Gly--Gly, or any other linker comprised in the art.
  • the TNF-induced septic shock effect may be assayed by means of the increase of the survival time after injections of the antibody to LPS-treated Balb/C mice as illustrated in the Examples section.
  • the tip region of TNF- ⁇ may be implicated in the toxic side effects of the cytokine on cells, thereby limiting the wider therapeutic use of TNF- ⁇ on normal cells (reviewed by Taniguchi and Sohmura, 1991). Therefore, antibodies recognizing the tip region of TNF- ⁇ can be of particular interest to discriminate between the tumoricidal effect of TNF- ⁇ on the one hand, and the trypanolytic and toxic effect of TNF- ⁇ on the other. Antibodies recognizing the above-mentioned TNF- ⁇ tip region polypeptides of the invention, will be further referred to as anti-tip antibodies.
  • the present invention relates also to the use of an antisense peptide of a peptide comprising at least part of the region extending from amino acids 101 to 116 of TNF- ⁇ , preferably comprising at least part of the region extending from amino acids 105 to 110 characterized in that it:
  • TNF- ⁇ inhibits the toxic effect of TNF- ⁇ with from 5 to 50%, preferably at least 10%, more preferably with at least 25%, most preferably with at least 35%; or,
  • antisense peptide is reviewed by Blalock (1990) and by Roubos (1990).
  • the molecular recognition theory (Blalock, 1990) states that not only the complementary nucleic acid sequences interact but that, in addition, interacting sites in proteins are composed of complementary amino acid sequences (sense ligand with receptor or sense ligand with antisense peptides).
  • two peptides derived from complementary nucleic acid sequences in the same reading frame will show a total interchange of their hydrophobic and hydrophilic amino acids when the amino terminus of one is aligned with the carboxy terminus of the other. This inverted hydropathic pattern might allow two such peptides to assume complementary conformations responsible for specific interaction.
  • the antisense peptides can be prepared as described in Ghiso et al. (1990). By means of this technology it is possible to logically construct a peptide having a physiologically relevant interaction with a known peptide by simple nucleotide sequence analysis for complementarity, and synthesis of the peptide complementary to the binding site.
  • the antisense peptides according of this preferred embodiment of the invention can be prepared by classical chemical synthesis.
  • the present invention relates to the use of any of the above-defined antibodies or antisense peptides, characterized in that they increase the half life time of TNF- ⁇ .
  • Antibodies or antisense peptides according to this latter embodiment are preferably used in combination with TNF- ⁇ .
  • TNF muteins according to the present invention can be administered either directly in the form of mutein protein in a pharmaceutical composition as described above, or in the form of cells transfected with the nucleic acid coding for such muteins, said nucleic acid being inserted into any suitable vector, as known to those skilled in the art.
  • the present invention relates also to a method for treating patients suffering from any of the above mentioned illnesses consisting of administering cells transfected with the cDNA coding for the TNF muteins as defined above, said nucleic acid being inserted into any suitable vector, as known to those skilled in the art.
  • the cells transfected by such a vector-nucleic acid combination are preferably autologous cells derived from the patient (e.g. a cancer patient) to be treated with such compositions.
  • Said vector-nucleic acid combination has to be constructed in such a way as to allow continuous expression of the mutein protein at either a constant level or at a level which can be modified, depending on the exact nature of the vector used to make the vector-nucleic acid combination.
  • the present invention may be used to treat cancer patients.
  • TNF- ⁇ tumor necrosis factor-alpha
  • mTNF- ⁇ mouse tumor necrosis factor-alpha
  • hTNF- ⁇ human tumor necrosis factor-alpha
  • FIGS. 3A and 3B In vitro trypanocidal activity of recombinant mouse (m) or human (h) TNF- ⁇ on T. brucei brucei (A) and T. rhodesiense (B).
  • m recombinant mouse
  • h human TNF- ⁇ on T. brucei brucei
  • T. rhodesiense B
  • 2 ⁇ 10 6 /ml purified bloodstream trypanosomes isolated 1 day before the first in vivo maximum peak, are incubated for 5 hours in PBS, 1% glucose, 1% normal mouse serum at 37° C. with various concentrations of recombinant TNF- ⁇ .
  • the number of living parasites is assessed and is compared with the control wells in which only incubation medium was added.
  • the spontaneous mortality in the control wells was always lower than 10%.
  • the values represent the means of quadruplicates and the standard deviation.
  • FIG. 4 LiCl potentiates the mouse TNF- ⁇ mediated trypanolysis in vitro. Trypanosomes were incubated for 5 hours with serial dilution of mTNF- ⁇ alone or enriched with 1 ⁇ g/ml of LiCl either in the presence or absence of anti-mTNF- ⁇ 1F3F3D4 monoclonal antibodies, which are capable of neutralizing the cytotoxic effect of TNF- ⁇ . The values represent the means of quadruplicates and the standard deviatation.
  • the values in the LiCl group differed significantly ( ⁇ 0.0008 and 0.009) from the control group indicating that LiCl synergistically potentiates the trypanocidal effect of mTNF- ⁇ . LiCl by itself had no effect on the survival of the trypanosomes as no difference with control values were seen (data not shown).
  • FIGS. 6A and 6B Preincubation of mTNF- ⁇ with N,N'-diacetylchitobiose (indicated as chitobiose) potently inhibits the trypanocidal activity (A), but not the cytolytic activity (B) of mouse TNF- ⁇ .
  • mTNF- ⁇ was preincubated for 2 h with 1 ⁇ g/ml of N,N'-diacetylchitobiose before adding it in a serial dilution to bloodsteam forms of T. brucei brucei (A) or to L929 cells (B) to be tested on its trypanocidal or cytolytic activity (as described in the examples), respectively.
  • FIG. 7 Trypanocidal activity of the circularized, biotinylated mouse TNF- ⁇ tip peptide (Bio- GG-CGPKDTPEGAELKPWYC) (mtip) and the biotinylated human TNF- ⁇ tip peptide (Bio-GG-CGQRETPEGAEAKPWYC) (htip), and the biotinylated linear subpep1 (Bio-GG-CTPGAEC) (subtip) on the bloodstream forms of T. brucei brucei and inhibition of the trypanocidal activity by pre-incubation with N,N'-diacetylchitobiose (Chitobiose).
  • FIG. 8 Trypanocidal activity of different synthetic tip peptides.
  • FIG. 9 Trypanocidal activity of subpeptide 1 and muteins thereof.
  • Serial dilutions of subpep 1 (Bio-GG-C-TPEGAE-C), subpep 3 (Bio-GG-TPE), mutpep 1 (Bio-GG-C- ⁇ PEGAE-C), mutpep 2 (Bio-GG-C-TAEGAE-C), mutpep 3 (Bio-GG-C-TPAGAE-C), mutpep 4 (Bio-GG-C-TPEAAE-C) and mutpep 5 (Bio-GG-C-TPEGAA-C) made in 100 ⁇ l were added to 2 ⁇ 10 5 /ml purified bloodstream forms of T. brucei brucei (final concentration 10 6 parasites/ml). As in FIG. 8, after 5 hours, the number of live parasites is assessed and compared with the control wells in which only incubation medium is added.
  • FIG. 10 The anti-mTNF- ⁇ tip polyclonal antibody (anti-tip) potently inhibits the trypanocidal effect of mTNF- ⁇ , but it only weakly inhibits the cytolytical activity against L929 cells.
  • mTNF- ⁇ was preincubated for 1 h with 10 ⁇ g/ml of protein G purified anti-tip polyclonal IgG before adding it to 2 ⁇ 10 6 bloodstream purified T. brucei brucei/ml. After 5 hours the trypanocidal activity of mTNF- ⁇ alone or pretreated with anti-tip polyclonal IgG was evaluated by counting the number of live parasites.
  • the cytolytical activity of TNF- ⁇ alone or pretreated with anti-tip polyclonal IgG was evaluated on cell survival of L929 cells after 48 hours incubation in the absence of actinomycin D.
  • FIG. 11 In vitro cytolytic (cytotoxic) activity of wild type mTNF- ⁇ and mTNFT105A on L929 cells as tested in the 48 hours assay in the absence of Actinomycin D. The assay was performed as explained in the Materials and Methods section of the Examples.
  • the soluble p55 hTNF- ⁇ receptor protein was obtained from Dr. D. Wallach (Dept. of Membrane Research & Biophysics, Weizmann Institute, Rehevot, Israel).
  • N,N'-diacetylchitobiose was purchased from Sigma (St. Louis, Mo., USA).
  • the TNF- ⁇ -derived tip peptides are preincubated for 1 h with 1 ⁇ g/ml oligosaccharide.
  • the peptide sequences are given left to right which, by convention, is the direction from the amino terminus to the carboxyterminus.
  • This new 727 bp sequence was then ligated into the SspI/XbaI opened vector pIG2 (SEQ ID NO 2), resulting in the vector pIG2mTNF.
  • pIG2 is a versatile expression vector for E. coli. It contains the temperature-inducible leftward promotor of phage lambda, a synthetic ribosome binding site, a multilinker sequence, the rrnBT1T2 transcriptional terminator and a tetracycline resistance gene (FIGS. 1 and 2)
  • Transformation and subsequent induction of pIG2mTNF to E. coli host MC1061(pAcI) resulted in an expression level of ⁇ 10% of total cellular protein (data not shown).
  • the mTNF- ⁇ Thr105 mutant wherein the Thr105 was replaced by an Ala residue, was generated by introducing the synthetic oligonucleotide primers 5'-CAAGGACGCTCCGGAGGGGGCTGAGCT-3' and 5'-CAGCCCCCTCCGGAGCGTC-3' in the StyI-SacI digested wild type (wt) mTNF- ⁇ , thereby replacing the Thr105 by an Ala residue and creating an unique BstEI control restriction site in the mutated TNF insert, which is not present in the wt TNF- ⁇ sequence.
  • the digested DNA was purified via the Geneclean method (Bio101, La Jolla) and resuspended in 68 ⁇ l of sterile doubledistilled water.
  • a 911 bp BamHI/StyI fragment was isolated from a 1.2% agarose gel via the Geneclean method and resuspended in 20 ⁇ l sterile doubledistilled water. This material is called fragment 1.
  • the digested DNA was purified via the Geneclean method (Bio101, La Jolla) and resuspended in 68 ⁇ l of sterile doubledistilled water.
  • a 3071 bp SacI/BamHII fragment was isolated from a 1.2% agarose gel via the gene clean method and resuspended in 40 ⁇ l sterile doubledistilled water. This material is called fragment 2.
  • Fragment 1 (5 ⁇ l), fragment 2 (1.5 ⁇ l) and the annealed oligos (10 ⁇ l) were mixed in a total volume of 19 ⁇ l ligation buffer (Boehringer Mannheim), 1 Units of T4 ligase (Boehringer Mannheim, 1 Unit/ ⁇ l) was added and the mixture was incubated for 3 hours at 15° C.
  • the ligation mixture was transformed to DH1(lambda) competent cells, plated out on Tetracyclin containing LB plates and grown overnight at 37° C. Plasmid DNA was prepared from selected colonies and analysed via restriction digestion (BspEI, BamHI, StyI and SacI). A via restriction analysis confirmed vector pIG2mTNFT105A was transformed to MC1061 (pAcI). This vector/host combination was further used for prokaryotic expression of the mTNFT105A mutein.
  • the digested DNA was purified via the Geneclean method (Bio101, La Jolla) and resuspended in 68 ⁇ l of sterile doubledistilled water.
  • a 911 bp BamHI/StyI fragment was isolated from a 1.2% agarose gel via the Geneclean method and resuspended in 20 ⁇ l sterile doubledistilled water. This material was called fragment 3.
  • the digested DNA was purified via the Geneclean method (Bio101, La Jolla) and resuspended in 68 ⁇ l of sterile doubledistilled water. To this volume 8 ⁇ l of 10 times concentrated buffer A (Boehringer Mannheim) and 40 Units SacI (Boehringer Mannheim, 10 U/ ⁇ l) were added and again incubated for 3 hours at 37° C.
  • a 3071 bp SacI/BamHII fragment was isolated from a 1.2% agarose gel via the Geneclean method and resuspended in 40 ⁇ l sterile doubledistilled water. This material was called fragment 4.
  • fragment 2 3071 bp SacI blunt/BamHI fragment from pIG2mTNF
  • aminocapronic acid, PMSF and DTT are added to a final concentration of respectively 25 mM, 1 mM and 1 mM before lysing by French Press (14,000 psi, AMINCO, SLM Instruments, Urbana, USA). Subsequently, the suspension is centrifugated for 20 minutes at 16,500 rpm at 4° C. using a JA-20 rotor (Beckman). Wild-type mTNF- ⁇ is found in the supernatant fraction as assessed by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE). Some of the TNF- ⁇ tip muteins were retrieved in the soluble and insoluble fraction.
  • the described procedure is directed to the recovery of the mTNF- ⁇ material out of the soluble fraction.
  • No attemps were made to purify and renaturate mTNF from the insoluble fraction.
  • the nucleic acids in the supernatant were precipitated at pH 7.0 by the addition of polyethylene-imine to a final concentration of 0.4% followed by centrifugation by a JA-20 rotor for 20 minutes at 16,500 rpm at 4° C.
  • mTNF- ⁇ or mutein protein were then enriched by a differential amoniumsulphate precipitation whereby a 40% step is followed by a second step at 65% saturation.
  • mTNF- ⁇ or muteins remained in the 65% amonium sulphate pellet.
  • the pellet was resuspended in 25 mM Tris-HCl pH 7.2 and the conductivity of the solution was adjusted to 10 mS above the conductivity of the Phenyl sepharose column startbuffer (25 mM TrisHCl pH 6.9, 25% (NH4)2SO4).
  • the material was then loaded at 0.5 cm/minute on the Phenyl Sepharose 4B matrix (Pharmacia) and washed with 8 column volumes of starting buffer. Proteins bound to the matrix were eluted with a linear salt gradient of 0% to 60% buffer B (20 mM ethanolamine, 10% ethylene glycol pH 9.0) over 6 column volumes.
  • the conductivity of the supernatant was adjusted to 10 mS above this of the equilibration buffer of the Phenyl Sepharose 4B column.
  • the chromatographical conditions were identical to those described in section 7.1, except that an equivalent of a 5 liter cell culture was loaded on a 40 ml column.
  • the T105A mTNF- ⁇ mutein was recovered in the first part of the gradient (about 0.9M ⁇ 0,5M (NH4)2SO4).
  • the pools of the 3 runs on Phenyl Sepharose 4B were mixed and after adjustment of the pH and the conductivity, the total pool was loaded on a 16 ml Phenyl Q Sepharose FF column. Chromatography conditions (flow rate (cm/h) and slope of the gradient) were kept identical as for these used on the previous Phenyl Sepharose 4B column.
  • the non-bound protein fraction was dialysed against 25 mM Tris-HCl, pH 9.0 and applied at 1 cm/min on a Q Sepharose FF column (Pharmacia) (>5 mg protein/ml gel). Bound proteins were eluted at 1 cm/min by applying a linear salt gradient (0-300 mM NaCl), followed by a 300 mM NaCl wash (2 column volumes). SDS-PAGE shows that the del mTNF- ⁇ protein was found all over the eluate, but only the fractions eluting between 70-230 mM NaCl were pooled. These fractions were lacking the contaminating E. coli proteins with a molecular weight of about 14000, 15000 and 70000.
  • the pooled fractions were diluted with the 25 mM Tris-HCl, pH 9.0 buffer and incubated for about 4 h at 4° C. Thereafter, the del mTNF- ⁇ pool was reloaded on the Q-sepharose FF column but now at a concentration ⁇ 300 ⁇ g protein/ml gel (protein quantity based on absorbance at 280 nm).
  • the wash and elution conditions were identical to these described for the first chromatography on Q Sepharose.
  • the tip-deleted mTNF- ⁇ protein eluted in 2 well separated peaks with pure del mTNF- ⁇ protein in the second peak (elution at about 280 mM NaCl).
  • the material has a specific activity of about 1 ⁇ 108 U/mg of protein as assessed by bioassay on L-929 cells in the presence of Actinomycin D.
  • aminocapronic acid, PMSF and DDT weree added to a final concentration of respectively 25 mM, 1 mM and 1 mM before lysing by French Press (14,000 psi, AMINCO, SLM Instruments, Urbana, USA). Subsequently, the suspension was centrifugated for 20 minutes at 16,500 rpm at 4° C. using a JA-20 rotor (Beckman). The mTNF- ⁇ double mutant could be found back in the supernatant fraction as assessed by SDS-PAGE and therefore only this fraction was used for further purification.
  • the nucleic acids in the supernatant were precipitated by the addition of polyethylene-immine to a final concentration of 0.4% followed by centrifugation for 20 minutes at 16,500 rpm at 4° C.
  • the mTNF- ⁇ double mutant precipitated only partially after polyethylene imine treatment and was recovered from the pellet by introducing a washing step with 0.5 ml NaCl pH 7.2. Both fractions were then enriched by a differential amoniumsulphate precipitation whereby a 40% step is followed by a second step at 65% saturation.
  • the mTNF- ⁇ mutein remained in the 65% amonium sulphate pellet.
  • the pellet was resuspended in 25 mM Tris-HCl pH 7.2 and the conductivity of the solution was adjusted to 10 mS above the conductivity of the Phenyl Sepharose 4B column startbuffer (25 mM TrisHCl pH 6.9, 25% (NH4)2SO4).
  • the material was loaded at 0.5 cm/minute on the Phenyl Sepharose 4B matrix (Pharmacia) and washed with 8 column volumes of starting buffer. Proteins bound to the matrix were eluted with a linear salt gradient of 0% to 60% buffer B (20 mM ethanolamine, pH 9.0) over 6 column volumes.
  • the flow through fractions containing the mTNF- ⁇ double mutant protein were pooled and dialysed overnight against 25 mM Tris-HCl pH 7.2. After centrifugation, the material was loaded at 1 cm/minute on Q Sepharose FF (Pharmacia) equilibrated against 25 mM Tris-HCl pH 7.2. The column was washed with 4 column volumes of starting buffer and eluted with a linear salt gradient from 0 mM to 300 mM NaCl in the same buffer followed by stepwise elution with 2 column volumes of 300 mM NaCl and 2 column volumes of 1 mM NaCl. The mTNF- ⁇ double mutant eluted at 300 mM NaCl.
  • the pellet was resuspended in 25 mM Tris-HCl pH 7.2 and the conductivity of the solution was adjusted to 10 mS above the conductivity of the Phenyl Sepharose 4B column startbuffer (25 mM Tris-HCl pH 6.9, 25% (NH4)2SO4).
  • the material was loaded at 0.5 cm/minute on the Phenyl Sepharose 4B matrix (Pharmacia) and washed with 8 column volumes of starting buffer. Proteins bound to the matrix were eluted with a linear salt gradient of 0% to 60% buffer B (20 mM ethanolamine, pH 9.0) over 6 column volumes.
  • the fractions containing the mTNF- ⁇ triple mutant (this is the flow through and the 20 to 40% eluate fractions) were pooled and loaded on a TSK-C4 650M (Merck) at 1 cm/h (10 mg/ml) and washed with 8 column volumes of Phenyl Sepharose starting buffer.
  • the proteins bound to the matrix were eluted with a linear gradient of 10 column volumes from 0% B to 100% B (20 mM ethanolamine pH 9.0).
  • the material was then loaded on a Q Sepharose FF (Pharmacia) column equilibrated against 25 mM Tris-HCl pH 7.2 at 1 cm/minute.
  • the Trypanocidal Region of TNF- ⁇ Contains a Lectin-like Activity of the Molecule
  • Recombinant mTNF- ⁇ and hTNF- ⁇ are trypanocidal for T. brucei brucei and T. brucei rhodesiense and the effect is potentiated by cotreatement with LiCl
  • the biotinylated tip peptides inspired on the sequence spanning amino acid positions 100 to 116 of hTNF- ⁇ (Pennica et al., 1984) and mTNF- ⁇ (Fransen et al., 1985) were synthesized as indicated in the materials and methods section. Subsequently, the peptides were analyzed using a C18 reversed phase HPLC column. Analysis of the three-dimensional structure of the TNF- ⁇ molecule upon consulting the Brookhaven data base demonstrated that the identified epitope is located at the tip of the molecule. This location is interesting considering the fact that TNF- ⁇ was shown to be active in a membrane-bound form (Kriegler et al., 1988).
  • sequences are given in the one letter amino acid code.
  • the amino acids in the L or D configuration are anotated in the one letter code in capitals or in small letters, respectively.
  • the annotation Bio-GG- denotes Biotin-Gly-Gly.
  • the synthetic mTNF- ⁇ tip peptide is directly trypanocidal in vitro, an effect that can be inhibited by preincubation of the peptide with N,N'-diacetylchitobiose
  • hTNF- ⁇ -tip peptide shows similar results (FIG. 7).
  • h/mTNF- ⁇ has a specific activity of 2 ⁇ 10 5 U/mg of protein, while the peptide exerts its activity with an average specificity of 4 ⁇ 10 2 -4 ⁇ 10 3 U/mg of peptide.
  • the trypanocidal activity of the peptide can also be inhibited by preincubating it for 1 h with 1 ⁇ g/ml of N,N'-diacetylchitobiose (FIG. 7), demonstrating that the tip region of h/mTNF- ⁇ (from amino acid position 100 to 116) is involved in its trypanocidal and its lectin-like activity.
  • the short tip peptide, Thr Pro Glu Gly Ala Glu contains the trypanocidal activity of TNF- ⁇ and is linked with the described lectin-like activity of the protein since it can be inhibited by preincubation with N,N'-diacetylchitobiose
  • the trypanocidal activity of the different mutant peptides 1, 2, 3, 4 and 5 of h/mTNF- ⁇ was measured, wherein positions 105, 106, 107, 108 or 110 of the TPEGAE peptide were replaced consecutively by an A.
  • the mutant peptides in which the T(105) or E(107) or E(110) was replaced by an A lost their trypanocidal activity, while the two other mutants were fully or even more active (FIG. 8 and 9).
  • Polyclonal antibodies against the mTNF- ⁇ and hTNF- ⁇ -derived tip peptides were produced as follows: On day 0, rabbits were injected subcutaneously with 50 ⁇ g of a complex existing of 7 ⁇ g of the biotinylated tip peptide and 43 ⁇ g of avidin (Neutralite, Eurogentec, Belgium) dissolved in complete Freund's adjuvant (total volume: 1 ml) (Difco Laboratories, Detroit, Mich., USA). On day 21, rabbits were injected subcutaneously with 50 ⁇ g of the same complex dissolved in incomplete Freund's adjuvant (total volume: 1 ml).
  • the rabbits were injected subcutaneously with 100 ⁇ g of the avidin-tip peptide complex in 1 ml of PBS.
  • the rabbits were bled, and the sera which reacted positively in ELISA with both the synthetic h/mTNF- ⁇ tip peptide bound to streptavidin and with the native h/mTNF- ⁇ molecule, were purified on Protein G Sepharose CL4B (Sigma, St. Louis, Mo., USA) essentially as proposed by the supplier.
  • the IgG concentration was assessed by the Bradford assay.
  • mice were immunized with 100 ⁇ g of avidine-biotine-TNF- ⁇ tip peptide conjugates emulsified in complete Freund's adjuvant. Three weeks later the same animals were challenged with 400 ⁇ g of avidin. Biotin-TNF- ⁇ tip peptide conjugates emulsified in incomplete Freund's adjuvant. Another three weeks later, the mice were boosted with 10 ⁇ g of avidin-biotin-TNF- ⁇ tip peptide conjugates dissolved in PBS. Three days later the spleen cells were fused to generate monoclonal antibodies.
  • Anti-mTNF- ⁇ tip polyclonal antibodies can inhibit LPS-induced septic shock in vivo
  • TNF- ⁇ has a lectin-like affinity for the glycoprotein uromodulin.
  • this glycoprotein is able to potently inhibit the LPS-induced shock in vivo, whereas it does not block the tumoricidal activity of TNF- ⁇ on L929 cells in vitro.
  • mice Since the TNF- ⁇ tip region is involved in the lectin-like activity of TNF- ⁇ , as demonstrated in the previous sections, the effect of the rabbit anti-mTNF- ⁇ tip polyclonal antibodies on LPS-induced septic shock in vivo was investigated.
  • Table V intraperitoneal injection of Balb/C mice (10 mice/group) with 100 ⁇ g of protein G-purified anti-tip polyclonal antibodies, 2 h before the injection of the lethal dose of 500 ⁇ g of LPS/mouse, resulted in an increased survival time as compared with the control mice that only received PBS or an irrelevant control polyclonal antibody preinjection (control IgG).
  • Recombinant and mutated mTNF- ⁇ and hTNF- ⁇ are cytotoxic to L-929 cells in vitro.
  • T(105), E(107), and/or E(110) mutated or tip deleted TNF- ⁇ has lost the mTNF- ⁇ trypanocidal activity.
  • T(105), E(107), and/or E(110) mutants, combinations of these and deletions of the TPEGAE human or mouse TNF- ⁇ tip domain result in a significant loss of mTNF- ⁇ trypanocidal activity (FIG. 12). It is to be remarked in this context that, mutation of the P(106) and/or G(108) in the tip peptide to A did not change the trypanocidal nor the cytolytic activity of TNF- ⁇ (data not shown).
  • T(105), E(107) and/or E(110) tip-mutated or tip-deleted mTNF- ⁇ has a lower LD50 value then wild type mTNF- ⁇ .
  • hMVECb Human microvascular endothelial cells of the brain (hMVECb) are prepared as described by Grau and Lou (1993).
  • the metastasis-promoting effect of wild type and tip deleted mouse TNF- ⁇ was compared by use of the 3LL-R tumorigenic Lewis lung carcinoma cell line (wt and del mTNF-resistant Lewis lung carcinoma) (Remels et al., 1989).
  • 3LL-R tumorigenic Lewis lung carcinoma cell line wt and del mTNF-resistant Lewis lung carcinoma
  • PBS control group
  • 5 ⁇ g of wild type or tip-deleted mouse TNF- ⁇ per mouse 5 hours prior to the intravenous injection of 2.106 3LL-R cells.
  • This tumor inoculum normally leads to a 100% mortality 25 days after injection of the control group.
  • mice injected with wt mTNF/3LL-R all suffered from severe respiration problems whereas the mice in the del mTNF/3LL-R group looked significantly better and the mice in the PBS/3LL-R group looked completely healthy.
  • all mice were killed and the lung weight as well as the amount of cancer cell nodules in the lung was assessed.
  • Table X The results are shown in Table X below.
  • the weight of the lungs as well as the number of nodules present in the lungs was significantly higher (at least 100 ⁇ ) in the TNF- ⁇ treated mice indicating that TNF- ⁇ indeed is capable of enhancing the metastatic potential of TNF-resistant tumor cells.
  • the metastatic enhancing potential of the TNF tip deletion mutant lays considerably lower than that of wild type TNF.
  • Lymphotoxin is expressed as a heteromeric complex with a distinct 33-kDa glycoprotein on the surface of an activated human T-cell hybridoma. J Biol Chem, 267:2542-2547.
  • Tumor necrosis factor ⁇ restores granulomas and induces parasite egg-laying in schistosome-infected SCID mice. Nature 356:604-607.
  • Uromodulin Trop-Horsfall glycoprotein: a renal ligand for lymphokines. Science 237:1479-1484.
  • TNF- ⁇ Tumour necrosis factor
  • TNF Tumor necrosis factor
  • Tumor necrosis factor plays a protective role in experimental cutaneous leishmaniasis. J Exp Med 170:2097-2104.

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US6346247B1 (en) 1999-10-28 2002-02-12 Promega Corporation Prevention and treatment of autoimmune disease with luminally administered polyclonal antibodies
WO2002040675A1 (fr) * 2000-11-15 2002-05-23 Shanghai Research Center Of Biotechnology, Chinese Academy Of Sciences Nouvelle proteine mutante du facteur onconecrosant humain et leur procede de preparation et application
JP3344609B2 (ja) 1995-01-09 2002-11-11 渡邊 定治 変異型ヒト腫瘍壊死因子
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JP3344609B2 (ja) 1995-01-09 2002-11-11 渡邊 定治 変異型ヒト腫瘍壊死因子
US20030185791A1 (en) * 1998-08-14 2003-10-02 Rudolf Lucas TNF-derived peptides for use in treating oedema
US7258861B2 (en) 1998-08-14 2007-08-21 Innogenetics N.V. TNF-derived peptides for use in treating oedema
US6346247B1 (en) 1999-10-28 2002-02-12 Promega Corporation Prevention and treatment of autoimmune disease with luminally administered polyclonal antibodies
WO2002040675A1 (fr) * 2000-11-15 2002-05-23 Shanghai Research Center Of Biotechnology, Chinese Academy Of Sciences Nouvelle proteine mutante du facteur onconecrosant humain et leur procede de preparation et application
US20050049402A1 (en) * 2002-12-02 2005-03-03 Babcook John S. Antibodies directed to tumor necrosis factor and uses thereof
US7285269B2 (en) 2002-12-02 2007-10-23 Amgen Fremont, Inc. Antibodies directed to tumor necrosis factor
US20080187531A1 (en) * 2002-12-02 2008-08-07 Babcook John S Antibodies directed to tumor necrosis factor and uses thereof
US8101178B2 (en) 2002-12-02 2012-01-24 Amgen Fremont Inc. Antibodies directed to tumor necrosis factor and uses thereof
WO2005099747A1 (fr) * 2004-04-14 2005-10-27 Medical Research Council Elimination selective de cellules cancereuses par induction d'acetyltransferase via des cytokines tnf-alpha et il-6
US20070207120A1 (en) * 2004-04-14 2007-09-06 Sarah Drayton Selective Killing Of Cancer Cells By Induction Of Acetyltransferase Via Tnf-Alpha And Il-6

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